Search

Fluorescent RNA aptamers are tools for studying RNA localization and interactions in vivo. The photophysical properties of these in vitro selected RNAs should be characterized prior to cellular imaging experiments. Here, we describe the process of determining the fluorophore affinity, fluorescence enhancement, and fluorescence lifetime(s) of the Mango-III fluorescence turn-on aptamer. Parameters determined through these protocols will aid in establishing conditions for live-cell imaging., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Oxygen (O 2 ) is a critical metabolite for cellular function as it fuels aerobic cellular metabolism; further, it is a known regulator of gene expression. Monitoring oxygenation within cells and organelles can provide valuable insights into how O 2 , or lack thereof, both influences and responds to cell processes. In recent years, fluorescence lifetime imaging microscopy (FLIM) has been used to track several probe concentration independent intracellular phenomena, such as pH, viscosity, and, in conjunction with Förster resonance energy transfer (FRET), protein-protein interactions. Here, we describe methods for synthesizing and expressing the novel FLIM-FRET intracellular O 2 probe Myoglobin-mCherry (Myo-mCherry) in cultured cell lines, as well as acquiring FLIM images using a laser scanning confocal microscope configured for two-photon excitation and a time-correlated single photon counting (TCSPC) module. Finally, we provide step-by-step protocols for FLIM analysis of Myo-mCherry using the commercial software SPCImage and conversion of fluorescence lifetime values in each pixel to apparent intracellular oxygen partial pressures (pO 2 ).

As the other chapters attest, sensitivity of fluorescent molecules to their local environment has created powerful tools in the study of molecular biology, particularly in the study of protein, DNA, and lipid dynamics. Surprisingly, even events faster than the nanosecond lifetimes of fluorophores are important in protein function, and in particular, events lasting just a few ps reflect on water motion and the coupled dynamics of proteins. These ultrafast phenomena can best be studied by using the same laser that excites fluorescence to also "strobe" the emission, providing sub-picosecond time slices of the action. We explain the strobing "upconversion" technique and some limits on its execution.